The materials described in this invention belong to a class of materials known as photoactivatable or "caged" probes. The term "cage" refers to a photolytically sensitive substituent that is designed to maximally interfere with the reactivity or other physical properties of the free probe. Flash photolysis of the "caged" probe causes an intramolecular reaction of the substituent, and releases the free, or uncaged, probe. Because the appearance of the free probe can be so carefully controlled, caged probes provide a means of controlling the release--both spatially and temporally--of the active product or reagent.
When the probe that is bound to a photoreactive group is highly colored or fluorescent, the blocked probe is known as a "caged" dye. Generally caged dyes are useful in that only the blocked precursor is present until illumination, whereupon the active dye or marker is produced. In the case of caged fluorescent dyes, the blocked precursors are generally non-fluorescent, slightly fluorescent, or have fluorescence that occurs at substantially shorter wavelengths than that of the corresponding free dye. Illumination of the blocked precursor then liberates a pulse of highly fluorescent dye. The blocked precursor can be added to a sample or sample stream and allowed to reach the target area, but only upon photolysis is the desired fluorescent dye produced in the illuminated area. Following photolytic illumination, the subsequent migration, diffusion, photobleaching, or localization of the dye can be observed as a function of the time and location since photolysis.
The use of a chemical blocking group that is removable by photolysis has been widely used and described using a variety of materials. A typical blocking group used for this purpose is o-nitroarylmethine. Upon photolysis with light that includes wavelengths less than about 400 nm, the o-nitroarylmethine group is intramolecularly converted to a derivative of an o-nitrosophenone. Fluorophores that have been protected using the o-nitroarylmethine caging group include various hydroxy and amino derivatives of anthracenes, naphthalenes, coumarins, fluorescein, resorufin, and rhodamine. Known caged fluorophores are lipophilic dyes that are not water soluble, and are consequently not useful for following the flow of water and water-containing fluids. The use of these caged fluorescent dyes is therefore limited to organic media.
The compounds of the present invention are caged derivatives of hydroxypyrenesulfonic acids. Unlike most commonly used fluorophores, pyrenesulfonic acids are fluorescent dyes that possess high water solubility, in addition to high absorbance and high fluorescence quantum yield. These properties make hydroxypyrenesulfonic acid dyes ideal fluorescent dyes for use with biological materials, as well as other aqueous systems.
Chemically reactive fluorescent pyrenyloxysulfonic acids are described in U.S. Pat. No. 5,132,432 by Haugland et al. (1992). Derivatives of pyrenesulfonic acids containing long alkyl chains for use as lipid probes are described in U.S. Pat. No. 4,844,841 by Koller et al. (1989). Neither the Haugland nor the Koller patent describes pyrenesulfonic acids containing photoremovable blocking groups. Esters of hydroxypyrenesulfonic acids have been prepared wherein the ester substituent can be cleaved from the hydroxypyrenesulfonic acid dye by enzyme action (Woltbeis et al., ANAL. BlOCHEM., 129, 365 (1983)). It is known that attaching this type of blocking group (a phosphate or an ester of an aliphatic carboxylic acid) to the hydroxyl group of 8-hydroxypyrene-1,3,6-trisulfonic acid shifts the absorption and fluorescence properties of the dye to noticeably shorter wavelengths. These derivatives do not contain photocleavable blocking groups, and have only been used to assay for the activity of various hydrolases.
Caged dyes utilizing fluorophores other than pyrenes have been chemically attached to water-soluble materials such as proteins and dextrans to detect protein movement and assembly inside living cells and to follow water flow. The caged fluorophores of the present invention have the advantage of being intrinsically water soluble without further modification, having a higher yield of fluorescent product on a per weight basis, and also liberate highly water soluble fluorescent products. Additionally, in contrast to other caged fluorophores, the caged hydroxypyrenesulfonic acid dyes of the present invention are uncaged very quickly when photolytically illuminated, allowing very precise spatial and temporal control of the appearance of free fluorescent dye. For example, in order to generate the most fluorescent form of fluorescein from a typical caged fluorescein compound, photolysis of two individual caging moieties is required, whereas in the compounds of the present invention, only one caging moiety needs to be photolysed to generate maximal fluorescence.
The caged dyes of the present invention exhibit particular utility for the study of flow dynamics in aqueous systems. Generally, flow analysis and velocimetry have previously been performed by injecting a dye or marker into the water flow under study. These techniques can only reveal the most general information about the flow dynamics of such systems. The use of free fluorescent dyes allows the dye marker to be homogeneously dispersed throughout the flow stream, and flow data can be gathered by interrogating the water stream using excitation illumination, allowing a degree of accuracy in measuring flow and dynamics. The use of photoactivated fluorescent dyes, however, allows even greater control over the appearance of the active, uncaged, fluorescent dye that is used as a marker (or "tagging" agent). Nevertheless, even with caged fluoresceins (for example), the rate of uncaging upon photolysis illumination is slow enough to limit the accuracy of velocimetric data gathered using these probes. Because the photoactivated hydroxypyrenesulfonic acid dyes, in contrast, uncage very quickly upon photolysis illumination, the dyes of the present invention allow a fluorescent label to be generated within the water stream with precise control of both time zero and space zero (the exact time and place that fluorescent marker initially appears in the stream).
The use of a caged pyrenetrisulfonic acid dye of the present invention in the evaluation of hydrodynamic flow around a lifting surface is described in Lempert et al., Paper No. AIAA-93-0517, 31st Aerospace Sciences Meeting & Exhibit, Jan. 11-14, 1993, Reno Nev. A caged fluorescein-dextran conjugate is also described in the above paper as useful for flow tagging. The non-polar character of the fluorescein fluorophore requires the conjugation of the dye to a dextran to give it the necessary water solubility for this use. As the caged pyrenesulfonic acid is completely water soluble, it can be utilized at a concentration of 5 mg/L, while the caged fluorescein-dextran can only be utilized at a concentration of 2 mg/L. The lower concentration of fluorescein conjugate, combined with the greater mass of the conjugate due to the presence of the bulky dextran, results in much lower concentration of fluorescent moieties that can be formed following photolysis within the flow stream.